EP3712272A1 - Procédé de modulation de l'épissage de l'arn par induction d'une mutation de base au niveau d'un site d'épissage ou d'une substitution de base dans une région de polypyrimidine - Google Patents

Procédé de modulation de l'épissage de l'arn par induction d'une mutation de base au niveau d'un site d'épissage ou d'une substitution de base dans une région de polypyrimidine Download PDF

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EP3712272A1
EP3712272A1 EP18837474.8A EP18837474A EP3712272A1 EP 3712272 A1 EP3712272 A1 EP 3712272A1 EP 18837474 A EP18837474 A EP 18837474A EP 3712272 A1 EP3712272 A1 EP 3712272A1
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exon
sgrna
fusion protein
interest
amino acid
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EP3712272A4 (fr
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Xing Chang
Juanjuan YUAN
Yunqing Ma
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Shanghai Institutes for Biological Sciences SIBS of CAS
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Shanghai Institutes for Biological Sciences SIBS of CAS
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Definitions

  • the disclosure relates to a method for modulating RNA splicing by inducing base mutation at splice site or base substitution in polypyrimidine region.
  • the correct expression of eukaryotic genes requires the removal of introns in the pre-mRNA and the splicing of exons to form mature mRNA. More than 98% of introns are excised by a highly dynamic protein complex, the spliceosome.
  • the spliceosome consists of more than 150 small nuclear ribonucleoproteins (snRNPs), such as U1, U2, U4, U5, and U6.
  • the U1 snRNP recognizes the GU sequence at the 5' splice site of the intron, splicing factor 1 (SF1) binds to the bifurcation point of the intron, and the 35KD subunit of the U2 auxiliary factor (U2AF) binds to the AG sequence at the 3' splice site of the intron, and its 65KD subunit binds to the polypyrimidine region sequence to complete the exon recognition process; then U5 and U6 proteins catalyze the intron removal process by regulating RNA structure reconstruction and RNA-protein interaction.
  • the RNA splicing process plays an important role in the regulation of gene expression.
  • RNA splicing process can be a possible therapeutic target for these diseases.
  • ASO antisense oligonucleotides
  • Antisense oligonucleotides can bind to cis-acting elements of RNA (such as exonic splicing enhancers) to block splicing of exons, but the use of antisense oligonucleotides to regulate splicing requires careful design and strict screening, and also requires continuous administration during treatment. Meanwhile, the synthesis of the antisense oligonucleotides is time-consuming and very expensive. Therefore, there is a dire need to provide a one-time cure for these diseases.
  • RNA such as exonic splicing enhancers
  • RNA splicing of a gene of interest in a cell characterized in that the method includes expressing targeting cytosine deaminase in the cell to induce mutation of the 3' splice site AG of an intron of interest of the gene of interest in the cell to AA, or mutation of the 5' splice site GT of an intron of interest of the gene of interest in the cell to AT, or mutation of multiple Cs in the polypyrimidine region of an intron of interest of the gene of interest in the cell to Ts.
  • the targeting cytosine deaminase used in the methods described herein may be selected from the group consisting of:
  • the targeting cytosine deaminase is the fusion protein of a cytosine deaminase, or a fragment or mutant thereof retaining enzyme activity, and a Cas enzyme with helicase activity and partial or no nuclease activity, or the fusion protein of a cytosine deaminase, or a fragment or mutant thereof retaining enzyme activity, and a Cpf enzyme with helicase activity and partial or no nuclease activity; the method includes expressing the targeting cytosine deaminase and an sgRNA in the cell, wherein the sgRNA is specifically recognized by the Cas enzyme or Cpf enzyme and binds to the sequence having a splice site of an intron of interest of the gene of interest, or binds to the complementary sequence of a polypyrimidine region of interest.
  • the targeting cytosine deaminase is the fusion protein of a cytosine deaminase, or a fragment or mutant thereof retaining enzyme activity, and an Ago protein; the method includes a step of expressing in the cell the targeting cytosine deaminase and a gDNA recognized by the Ago protein.
  • a method of regulating RNA splicing of a gene of interest in a cell comprising a step of expressing in the cell (1) a fusion protein of a Cas protein with helicase activity and partial or no nuclease activity, and cytosine deaminase AID or a mutant thereof, and (2) an sgRNA; wherein, the Cas protein recognition region of the sgRNA is specifically recognized by the Cas protein, and the sgRNA binds to the sequence having a splice site of an intron of interest of the gene of interest, or binds to the complementary sequence of a polypyrimidine region of interest.
  • the sgRNA binds to the sequence having the 5' splice site of the intron of interest of the gene of interest, and the fusion protein mutates the GT at the 5' splice site to AT, thereby inducing exon skipping, activating alternative splice sites, inducing mutually exclusive exon switching or intron retention.
  • the sgRNA binds to the sequence having the 3' splice site of the intron of interest of the gene of interest, and the fusion protein mutates the AG at the 3' splice site to AA, thereby inducing exon skipping, activating alternative splice sites, inducing mutually exclusive exon switching or intron retention.
  • the sgRNA binds to the complementary sequence of the polypyrimidine region of interest, and induces the C at the polypyrimidine region to T, thereby enhancing exon inclusion.
  • RNA splicing of the gene of interest in the cell is regulated by transferring expression vector(s) of the fusion protein and the sgRNA into the cell.
  • the method further includes a step of simultaneously transferring an expression plasmid of Ugi.
  • the method further includes a step of simultaneously transferring expression plasmid(s) of a fusion protein of a nuclease-deficient or nuclease-partially-deficient Cas9 protein, AID or a mutant thereof, and an Ugi.
  • the fusion protein and AID, a fragment or a mutant thereof are as described in any part or any embodiment herein.
  • the cell of interest and the gene of interest are as described in any part or any embodiment herein.
  • a method for inducing exon skipping comprising a step of expressing in the cell (1) a fusion protein of a Cas protein with helicase activity and partial or no nuclease activity, cytosine deaminase AID or a mutant thereof, and an optional Ugi fusion protein, and (2) an sgRNA; wherein, the Cas protein recognition region of the sgRNA is specifically recognized by the Cas protein, and the sgRNA binds to the sequence having a splice site of an intron of interest of the gene of interest.
  • a method for activating alternative splice site(s) comprising a step of expressing in the cell (1) a fusion protein of a Cas protein with helicase activity and partial or no nuclease activity, cytosine deaminase AID or a mutant thereof, and an optional Ugi fusion protein, and (2) an sgRNA; wherein, the Cas protein recognition region of the sgRNA is specifically recognized by the Cas protein, and the sgRNA binds to the sequence having a splice site of an intron of interest of the gene of interest, wherein the intron of interest has alternative splice site(s) nearby.
  • a method for inducing mutually exclusive exon switching comprising a step of expressing in the cell (1) a fusion protein of a Cas protein with helicase activity and partial or no nuclease activity, cytosine deaminase AID or a mutant thereof, and optional an Ugi, and (2) an sgRNA; wherein, the Cas protein recognition region of the sgRNA is specifically recognized by the Cas protein, and the target binding region of the sgRNA comprises the sequence of a splice site of an intron of interest of the gene of interest, wherein the gene of interest is slected from a group consisting of PKMs.
  • a method for inducing intron retention comprising a step of expressing in the cell (1) a fusion protein of a Cas protein with helicase activity and partial or no nuclease activity, cytosine deaminase AID or a mutant thereof, and optional an Ugi fusion protein, and (2) an sgRNA; wherein, the Cas protein recognition region of the sgRNA is specifically recognized by the Cas protein, and the sgRNA comprises a splice site of the intron of interest, wherein the intron of interest is short in length ( ⁇ 150bp) and rich in G/C bases.
  • a method for enhancing exon inclusion comprising a step of expressing in the cell (1) a fusion protein of a Cas protein with helicase activity and partial or no nuclease activity, cytosine deaminase AID or a mutant thereof, and optional an Ugi, and (2) an sgRNA; wherein, the Cas protein recognition region of the sgRNA is specifically recognized by the Cas protein, and the sgRNA comprises the complementary sequence of the polypyrimidine region upstream of the exon of interest.
  • fusion protein that contains a Cas protein with helicase activity and partial or no nuclease activity and cytosine deaminase AID or a mutant thereof.
  • the fusion protein herein also contains Ugi.
  • a fusion protein for generating a point mutation in a cell, or for regulating RNA splicing of a gene of interest in a cell, or for inducing exon skipping, activating alternative splicing sites, inducing mutually exclusion exon switching, inducing intron retention, or enhancing exon inclusion in a cell of interest, wherein the fusion protein contains a Cas protein with helicase activity and partial or no nuclease activity and cytosine deaminase AID or a mutant thereof, and optional a linker sequence, a nuclear localization sequence, and Ugi.
  • Also provided herein is a method for treating a disease using the method for regulating RNA splicing described herein.
  • fusion protein described herein or its expression vector and the corresponding sgRNA or its expression vector in the preparation of a kit for regulating RNA splicing, as well as a kit comprising the fusion protein described herein or its expression and the corresponding sgRNA or its expression vector.
  • RNA splicing of the gene of interest in the cell can be regulated, so that to induce exon skipping, activate alternative splice sites, induce mutually exclusive exon switching, induce intron retention or enhance exon inclusion.
  • "Regulating" herein means to change the conventional splicing manner of the RNA.
  • the present disclosure can be implemented using targeting cytosine deaminase.
  • the targeting cytosine deaminase is constructed by fusing cytosine deaminase with a protein with a targeting effect.
  • cytosine deaminase refers to various enzymes with cytosine deaminase activity, including but not limited to enzymes of the APOBEC family, such as APOBEC-2, AID, APOBEC-3A, APOBEC-3B, APOBEC-3C, APOBEC-3DE, APOBEC-3G, APOBEC-3F, APOBEC-3H, APOBEC4, APOBEC1 and pmCDA1.
  • the cytosine deaminase suitable for use herein can be derived from any species, preferably mammalian, especially human cytosine deaminase.
  • the cytosine deaminase suitable for use herein is an activated cytosine deaminase, such as a human-derived activated cytosine deaminase.
  • the cytosine deaminases of the APOBEC family are RNA editing enzymes with a nuclear localization signal at the N-terminus and a nuclear export signal at the C-terminus. The catalytic domain of these enzymes is shared by the APOBEC family. Generally, the N-terminal structure is considered necessary for somatic hypermutation (SHM).
  • cytosine deaminases The function of cytosine deaminases is to deaminate cytosine and transform cytosine into uracil, and then DNA repairing can transform uracil into other bases. It should be understood that the cytosine deaminases well known in the art or fragments or mutants thereof that retain the biological activity of deaminating cytosine and converting cytosine into uracil can be used herein.
  • AID is used herein as the cytosine deaminase in the targeting cytosine deaminase.
  • Amino acid residues 9-26 of AID are nuclear localization (NLS) domain, especially amino acid residues 13-26, which are involved in DNA binding; amino acid residues 56-94 are catalytic domain; amino acid residues 109-182 are APOBEC-like domain; amino acid residues 193-198 are nuclear export (NES) domain; amino acid residues 39-42 interact with catenin-like protein 1 (CTNNBL1); and amino acid residues 113-123 are hotspot recognition loop.
  • NLS nuclear localization
  • NES nuclear export
  • the full-length AID (as shown in SEQ ID NO: 25, amino acids 1457-1654), or a fragment of AID can be used in this disclosure.
  • the fragment includes at least the NLS domain, the catalytic domain, and the APOBEC-like domain. Therefore, in certain embodiments, the fragment comprises at least amino acid residues 9-182 of AID (i.e., amino acid residues 1465-1638 of SEQ ID NO: 25). In other embodiments, the fragment comprises at least amino acid residues 1-182 of AID (i.e., amino acid residues 1457-1638 of SEQ ID NO: 25).
  • the AID fragment used herein consists of amino acid residues 1-182, amino acid residues 1-186, or amino acid residues 1-190. Therefore, in certain embodiments, the AID fragment used herein consists of amino acid residues 1457-1638 of SEQ ID NO: 25, amino acid residues 1457-1642 of SEQ ID NO: 25, or amino acid residues 1457-1646 of SEQ ID NO: 25.
  • a variant of AID that retains its cytosine deaminase activity i.e., the biological activity of deaminating cytosine and converting cytosine into uracil
  • such variants may have 1-10, such as 1-8, 1-5, or 1-3 amino acid variations, including amino acid deletions, substitutions, and mutations, with respect to the sequence of the wild-type AID.
  • these amino acid variations do not present in the above-mentioned NLS domain, catalytic domain, or APOBEC-like domain, or even if they occur in these domains, they do not affect the original biological functions of these domains.
  • these variations do not occur at the amino acid residue 24, 27, 38, 56, 58, 87, 90, 112, 140 of the AID amino acid sequence. In certain embodiments, these variations also do not occur within amino acids 39-42, amino acids 113-123. Thus, for example, variations can occur in amino acids 1-8, amino acids 28-37, amino acids 43-55 and/or amino acids 183-198. In certain embodiments, variations occur at amino acids 10, 82, and 156.
  • amino acids 10, 82, and 156 which may be K10E, T82I, and E156G
  • amino acid sequence of the exemplary AID mutant contains or consists of the amino acid sequence shown as residues 1447-1629 of SEQ ID NO: 31.
  • Examples of other AIDs, fragments or mutants thereof can refer to CN201710451424.3 , the entire contents of which are incorporated herein by reference.
  • the protein with a targeting effect may be a protein known in the art that can target a gene of interest in the cell genome, including but not limited to a TALEN protein that specifically recognizes the target sequence, a zinc finger protein that recognizes the target sequence by mutation, an Ago protein, a Cpf enzyme and a Cas enzyme.
  • TALEN protein that specifically recognizes the target sequence
  • zinc finger protein that recognizes the target sequence by mutation
  • Ago protein an Ago protein
  • Cpf enzyme and a Cas enzyme a protein known in the art that can target a gene of interest in the cell genome
  • This disclosure can be implemented using TALEN proteins, zinc finger proteins, Ago proteins, and Cpf enzymes and Cas enzymes, which are well known in the art.
  • the targeting cytosine deaminase suitable for use herein may be selected from the group consisting of:
  • Cpf enzymes When Cpf enzymes are used, it is preferable to use a Cpf enzyme in which nuclease activity is partially or completely absent but helicase activity retains.
  • the Cpf enzyme under the guidance of its recognized sgRNA, binds to the specific DNA sequence, allowing the cytosine deaminase fused thereto to perform the mutations described herein.
  • the Ago protein needs to bind to the specific DNA sequence under the guidance of its recognized gDNA.
  • the targeting cytosine deaminase AID-mediated gene mutation technology is used herein to mutate guanine to adenine at the splice site of the intron, specifically block the exon recognition process, and regulate the alternative splicing process of endogenous mRNA.
  • TAM technique herein uses a fusion protein of a Cas protein lacking nuclease activity and cytosine deaminase AID, an active fragment or a mutant thereof.
  • the fusion protein is recruited to the specific DNA sequence, wherein AID, active fragments or mutants thereof mutates guanine (G) into adenine (A), or mutates cytosine (C) into thymine (T).
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • Cas protein with endonuclease activity also called Cas enzyme
  • sgRNA the complex formed by the Cas protein with endonuclease activity (also called Cas enzyme) and its specifically recognized sgRNA is complementary paired with the template strand in the target DNA through the matching region (i.e., target binding region) of the sgRNA, and the double-stranded DNA is cut at a specific location by Cas.
  • Cas/sgRNA are used in this disclosure, that is, the Cas is localized to the desired location through the specific binding of the sgRNA to the target, where the AID or its active fragment or mutant in the fusion protein mutates guanine (G) to adenine (A), or cytosine (C) to thymine (T).
  • the Cas protein suitable for this disclosure having helicase activity and partial (only having DNA single-strand break ability) or no nuclease activity (no DNA double-strand break ability), especially those having helicase activity and partial or no endonuclease activity, can be derived from various Cas proteins well known in the art and variants thereof, including but not limited to Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx
  • Cas9 enzymes lacking nuclease activity toghether with its specifically recognized single-stranded sgRNA are used.
  • Cas9 enzymes may be Cas9 enzymes from different species, including but not limited to Cas9 from Streptococcus pyogenes (SpCas9), Cas9 from Staphylococcus aureus (SaCas9), and Cas9 from Streptococcus thermophilus (St1Cas9), etc.
  • Various variants of the Cas9 enzyme can be used, provided that the Cas9 enzyme can specifically recognize its sgRNA and lack nuclease activity.
  • Cas proteins lacking nuclease activity can be prepared by methods well known in the art. These methods include, but are not limited to, deleting the entire catalytic domain of the endonuclease in the Cas proteins, or mutating one or several amino acids in the catalytic domain, thereby producing Cas proteins lacking nuclease activity.
  • the mutation may be deletion or substitution of one or several (for example, 2 or more, 3 or more, 4 or more, 5 or more, 10 or more to the entire catalytic domain) amino acid residues, or insertion of one or several (e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, or 1-10, 1-15) new amino acids residues.
  • the two endonuclease catalytic domains RuvC1 and HNH can be mutated separately, e.g., the amino acid 10 asparagine of the enzyme (in RuvC1 domain) is mutated to alanine or other amino acids, the amino acid 841 histidine (in HNH domain) is mutated to alanine or other amino acids.
  • the Cas enzyme has no nuclease activity at all.
  • the amino acid sequence of the nuclease-activity-free Cas9 enzyme used herein is shown as residues 42-1452 of SEQ ID NO: 25.
  • the Cas enzyme used herein partially lacks nuclease activity, i.e., the Cas enzyme can cause DNA single-strand breaks.
  • a representative example of such Cas enzymes can be shown as amino acid residues 42-1419 of SEQ ID NO: 33.
  • the amino acid sequence of the Cas enzyme used herein is shown as residues 199-1566 of SEQ ID NO: 23, or is shown as residues 199-1262 of SEQ ID NO: 50. Examples of other Cas enzymes can refer to CN201710451424.3 , the entire contents of which are incorporated herein by reference.
  • the Cas/sgRNA complex's function requires a protospacer adjacent motif (PAM) in the non-template strand (3' to 5') of the DNA.
  • PAM protospacer adjacent motif
  • the corresponding PAMs of different Cas enzymes are not exactly the same.
  • the PAM for SpCas9 is NGG (SEQ ID NO: 34); the PAM for SaCas9 is NNGRR (SEQ ID NO: 35); the PAM for St1Cas9 is NNAGAA (SEQ ID NO: 36); wherein N is A, C, T or G, and R is G or A.
  • the PAM for SaCas9 is NNGRRT (SEQ ID NO: 37).
  • the PAM for SpCas9 is TGG (SEQ ID NO: 38); in certain preferred embodiments, the PAM for SaCas9 enzyme KKH mutant is NNNRRT (SEQ ID NO: 39); wherein, N is A, C, T or G, and R is G or A.
  • sgRNA contains two parts: target binding region and protein recognition region (such as Cas enzyme recognition region or Cpf enzyme recognition region).
  • the target binding region and the protein recognition region are usually connected in a 5' to 3' direction.
  • the length of the target binding region is usually 15 to 25 bases, more usually 18 to 22 bases, such as 20 bases.
  • the target binding region specifically binds to the template strand of DNA, thereby recruiting the fusion protein to a predetermined site.
  • the opposite complementary region of the sgRNA binding region on the DNA template strand is immediately adjacent to PAM, or separated from PAM by several bases (for example, within 10, or within 8, or within 5 bases).
  • the enzyme's PAM is determined according to the used splicing enzyme (such as Cas enzyme), and then the non-template strand of DNA is searched for a site that can be used as PAM, and then a fragment of 15bp-20bp in length, more usually 18bp-22bp in length, which is downstream from the PAM site of the non-template strand (3' to 5') and immediately adjacent to the PAM site or separated from the PAM site within 10bp (e.g., within 8bp or 5bp) serves as the sequence of the target binding region of sgRNA.
  • the protein recognition region of sgRNA is determined according to the used splicing enzyme, which is known by those skilled in the art.
  • the sequence of the target binding region of the sgRNA herein comprises the fragment of 15bp-20bp in length, more usually 18bp-22bp in length, downstream from the PAM site recognized by the selected splicing enzyme (such as Cas enzyme or Cpf enzyme) and immediately adjacent to the PAM site or separated from the PAM site within 10bp (e.g., within 8bp or 5bp); its protein recognition region is specifically recognized by the selected splicing enzyme.
  • the selected splicing enzyme such as Cas enzyme or Cpf enzyme
  • the purpose of this disclosure is to mutate guanine to adenine at the intron splice site, or mutate C to T in the polypyrimidine strand upstream of the 3' splice site, it should be considered whether a PAM sequence is present near the splice site, and the distance between the PAM sequence and the splice site(s), when designing an sgRNA for this disclosure. Therefore, in general, the sgRNA binds to the sequence containing the splice site(s) of the intron of interest of the gene of interest, or to the complementary sequence of the polypyrimidine region of interest. Alternatively, the target binding region of the sgRNA contains the complementary sequence of the splice site(s) of the intron of interest of the gene of interest, or contains the sequence of the polypyrimidine region of the intron of interest of the gene of interest.
  • the sgRNA can be prepared by conventional methods in the art, for example, synthesized by conventional chemical synthesis methods.
  • the sgRNA can also be transferred into cells via an expression vector, and expressed in the cells; or it can be introduced into animals/humans via adeno-associated viruses.
  • the expression vector of the sgRNA can be constructed using methods well known in the art.
  • sgRNA sequences or complementary sequences thereof are also provided herein, which include a target binding region and a protein recognition region, wherein the target binding region binds to a sequence containing a splice site of the intron of interest of the gene of interest, or to a complementary sequence of the polypyrimidine region of interest.
  • the target binding region is 15-25bp in length, such as 18-22bp, preferably 20bp.
  • the target binding region of the sgRNA binds to the sequence in DMD exon 50 having the 3' splice site; preferably, the target binding region of the sgRNA is as shown in SEQ ID NO: 17 or 51.
  • the targeting cytosine deaminase used herein is preferably a fusion protein of the aforementioned Cas enzyme and the aforementioned AID or fragments or mutants thereof.
  • the Cas enzyme is usually at the N-terminus of the amino acid sequence of the fusion protein, and the AID or its fragment or mutant is at the C-terminus.
  • the AID or its fragment or mutant can be at the N-terminus of the amino acid sequence of the fusion protein, and the Cas enzyme is at the C-terminus.
  • fusion proteins substantially formed by a Cas enzyme and AID or a fragment or mutant thereof are provided herein.
  • the fusion protein "substantially formed by " or similar references herein does not indicate that the fusion protein only contains Cas enzyme and AID or its fragment or mutant thereof.
  • the phrase should be understood that the fusion protein can only contain Cas enzyme and AID or its fragment or mutant thereof, or the fusion protein can further contain other parts that do not affect the targeting effect of the Cas enzyme and the function to mutate target sequence(s) by AID or its fragment or mutant thereof in the fusion protein.
  • Said other parts include but are not limited to various linker sequences, nuclear localization sequences, Ugi sequences, and amino acid sequences introduced into the fusion protein due to gene cloning process and/or to construct the fusion protein, to promote expression of the recombinant proteins, to obtain the recombinant proteins automatically secreted from the host cells, or to facilitate the detection and/or purification of the recombinant proteins, as described below.
  • Cas enzymes can be fused to AID or fragments or mutants thereof via linkers.
  • the linker may be a peptide of 3 to 25 residues, for example, a peptide of 3 to 15, 5 to 15, 10 to 20 residues. Suitable examples of the peptide linkers are well known in the art.
  • a linker contains one or more motifs that repeat in sequence, which usually contain Gly and/or Ser.
  • the motif may be SGGS (SEQ ID NO: 40), GSSGS (SEQ ID NO: 41), GGGS (SEQ ID NO: 42), GGGGS (SEQ ID NO: 43), SSSSG (SEQ ID NO: 44), GSGSA (SEQ ID NO: 45) and GGSGG (SEQ ID NO: 46).
  • the motifs are adjacent to each other in the linker sequence, with no amino acid residue inserted between the repeated motifs.
  • the linker sequence may comprise or consist of 1, 2, 3, 4 or 5 repeated motifs.
  • the linker sequence is a polyglycine linker sequence.
  • the number of glycine in the linker sequence is not particularly limited, but is usually 2-20, such as 2-15, 2-10, 2-8.
  • the linker can also contain other known amino acid residues, such as alanine (A), leucine (L), threonine (T), glutamic acid (E), phenylalanine (F), arginine (R), glutamine (Q), etc.
  • the linker sequence is XTEN, and its amino acid sequence is shown as amino acid residues 183-198 of SEQ ID NO:29.
  • Other exemplary linker sequences can be the linker sequences described in CN201710451424.3 , such as SEQ ID NO: 21-31 described therein.
  • the N-terminus or C-terminus of the fusion protein herein may futher contains one or more polypeptide fragments as protein labels.
  • Any suitable label can be used for this disclosure.
  • the labels may be FLAG, HA, HA1, c-Myc, Poly-His, Poly-Arg, Strep-TagII, AU1, EE, T7, 4A6, ⁇ , B, gE, and Ty1. These labels can be used to purify proteins.
  • the fusion protein herein may also contain a nuclear localization sequence (NLS).
  • Nucleus localization sequences known in the art derived from various sources and with various amino acid compositions can be used.
  • Such nuclear localization sequences include, but are not limited to: NLS from SV40 virus large T antigen; NLS from nucleoplasmic proteins, for example, nucleoplasmic protein bipartite NLS; NLS from c-myc; NLS from hRNPA1M9; sequences from IBB domain of importin-a; sequences from myoma T protein; sequences from mouse c-ab1IV; sequences from influenza virus NS1; sequences from hepatitis virus ⁇ antigen; sequences from mouse Mx1 protein; sequences from human poly(ADP-ribose) polymerase; and sequences from steroid hormone receptor (human) glucocorticoid; etc.
  • NLS nuclear localization sequence
  • NLS The amino acid sequences of these NLS sequences can be found in CN201710451424.3 as SEQ ID NO: 33-47.
  • sequence shown by amino acid residues 26-33 of SEQ ID NO: 25 is used herein as NLS.
  • the NLS can be located at the N-terminus, C-terminus of the fusion protein; it can also be located within the sequence of the fusion protein, such as located at the N-terminus and/or C-terminus of the Cas9 enzyme in the fusion protein, or located at the N-terminal and/or C-terminal of the AID or its fragment or mutant in the fusion protein.
  • the accumulation of the fusion protein disclosed herein in the nucleus can be detected by any suitable technique.
  • detection labels can be fused to the Cas enzyme so that the location of the fusion proteins within cells can be visualized when combined with methods of detecting nucleus location (e.g., a dye specific to the nucleus, such as DAPI).
  • 3*flag is used as a label herein, and the peptide sequence may be amino acid residues 1 to 23 of SEQ ID NO:25. It should be understood that, generally, if a label sequence is used, the label sequence is at the N-terminus of the fusion protein.
  • the label sequence can be directly connected to NLS, or may be connected via an appropriate linker sequence.
  • the NLS sequence may be directly connected to the Cas enzyme or AID or its fragment or mutant, or it may be connected to the Cas enzyme or AID or its fragment or mutant through an appropriate linker sequence.
  • the fusion protein herein consists of a Cas enzyme and a AID or its fragment or mutant.
  • the fusion protein herein is formed by connection of a Cas enzyme to a AID or its fragment or mutant via a linker.
  • the fusion protein herein consists of a NLS, a Cas enzyme, a AID or its fragment or mutant, and optionally a linker sequence between the Cas enzyme and the AID or its fragment or mutant.
  • the fusion protein herein may also contain a phage protein, such as UGI as an UNG inhibitor.
  • the amino acid sequence of an exemplary UGI may be amino acid residues 1576-1659 of SEQ ID NO: 23 of the present disclosure. Therefore, in certain embodiments, the fusion protein herein contains the Cas9 enzyme described herein, the AID or a fragment or mutant thereof, UGI and NLS described herein, or consists of these parts, optional linker(s) between them and optional amino acid sequence(s) for detection, isolation or purification.
  • the Ugi sequence may be located at the N-terminus, C-terminus of the fusion protein, or within the fusion protein, for example, located between the NLS sequence and the Cas enzyme or between the Cas enzyme and the AID or a fragment or mutant thereof.
  • the fusion protein herein contains or consists of, from the N-terminus to the C-terminus, AID or a fragment or mutant thereof, Cas enzyme, Ugi and NLS, or contains or consists of, from the N-terminus to the C-terminus, Cas enzyme, AID or a fragment or mutant thereof, Ugi and NLS; they can be connected by linker(s).
  • the fusion proteins disclosed in CN 201710451424.3 are used herein. More specifically, the amino acid sequence of the used fusion protein disclosed in this disclosure is SEQ ID NO: 25, 27, 29, 31, 33, 48, or 50, or amino acids 26-1654 of SEQ ID NO: 25, or amino acids 26-1638 of SEQ ID NO: 27, or amino acids 26-1629 of SEQ ID NO: 31, or amino acids 26-1638 of SEQ ID NO: 33, or amino acids 26-1629 of SEQ ID NO: 48. In certain embodiments, the fusion protein herein is shown by SEQ ID NO: 23 of the present disclosure.
  • An expression vector/plasmid expressing the above fusion protein and a vector/plasmid expressing the desired sgRNA can be constructed and transferred into cells of interest to regulate their RNA splicing by inducing mutations at the splice site(s) of the gene of interest.
  • the "expression vector” may be various bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenovirus, retrovirus, or other vectors well known in the art. Any plasmid or vector can be used, provided that it can replicate and be stable in the host.
  • An important charecterastic of an expression vector is that it usually contains an origin of replication, a promoter, a marker gene and a translation control element.
  • the expression vector may also include a ribosome binding site for translation initiation and a transcription terminator.
  • the polynucleotide sequences described herein are operably linked to appropriate promoters in the expression vectors, so that mRNA synthesis is directed by the promoters.
  • promoters are: the lac or trp promoter of E.coli; the PL promoter of phage ⁇ ; eukaryotic promoters including the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, LTRs of retroviruses, and other known promoters that can control gene expression in prokaryotic or eukaryotic cells or their viruses.
  • Marker genes can provide phenotypic traits for selection of transformed host cells, including but not limited to dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell, or tetracycline or ampicillin resistance for E.coli.
  • Enhancers are cis-acting factors of DNA, usually are about 10bp to 300bp, which act on the promoter to enhance gene transcription.
  • the fusion protein herein, its coding sequence or expression vector, and/or the sgRNA, its coding sequence or expression vector may be provided in the form of a composition.
  • the composition may contain the fusion protein herein and the sgRNA or the vector expressing the sgRNA, or may contain the vector expressing the fusion protein herein and the sgRNA or the vector expressing the sgRNA.
  • the fusion protein or its expression vector, or sgRNA or its expression vector may be provided as a mixture, or may be packaged separately.
  • the composition may be in the form of a solution or a lyophilized form.
  • the fusion protein in the composition is a fusion protein of the AID or a fragment or mutant thereof described herein and the Cas enzyme described herein.
  • kits containing the compositions described herein are kits containing the compositions described herein.
  • the fusion protein or its expression vector, or sgRNA or its expression vector may be packaged separately, or may be provided as a mixture.
  • the kit may further include, for example, reagents for transferring into cells the fusion protein or its expression vector and/or sgRNA or its expression vector, and instructions for the transfer.
  • the kit may also include instructions for implementing the various methods and uses described herein using the ingredients contained in the kit.
  • the kit also includes other reagents, such as reagents for PCR.
  • the fusion protein herein, its coding sequence or expression vector, and/or the sgRNA or its expression vector can be used to induce base mutations at a splice site of the gene of interest to regulate its RNA splicing. Therefore, provided herein is a method for inducing base mutation in a splice site of a gene of interest in a cell of interest, wherein the method comprises the step of expressing the fusion protein described herein in the cell, the method also comprises the step of expressing sgRNAs or gDNAs based on the expressed fusion protein.
  • the fusion protein described herein of the AID or a fragment or mutant thereof and the Cas enzyme, together with its recognized sgRNA are expressed in cells.
  • the fusion protein of a cytosine deaminase, or a fragment or mutant thereof retaining enzyme activity, and a TALEN protein that specifically recognizes a target sequence is expressed in cells.
  • the fusion protein of a cytosine deaminase, or a fragment or mutant thereof retaining enzyme activity, and a zinc finger protein that specifically recognizes a target sequence is expressed in cells.
  • the fusion protein of a cytosine deaminase or a fragment or mutant thereof retaining enzyme activity and a Cpf enzyme with helicase activity and partial or no nuclease activity, together with the sgRNA recognized by the Cpf enzyme are expressed in cells.
  • the fusion protein of a cytosine deaminase or a fragment or mutant thereof retaining enzyme activity and an Ago protein, together with the gDNA recognized by the Ago protein are expressed in cells.
  • cells of interest especially also include those in which a splice site of a gene of interest needs to be mutated to regulate its RNA splicing.
  • Such cells include prokaryotic cells and eukaryotic cells, such as plant cells, animal cells, microbial cells, and the like.
  • animal cells such as mammalian cells, rodent cells, including cells of humans, horses, cattles, sheeps, mice, rabbits, and the like.
  • Microbial cells include cells from various microbial species that are well known in the art, especially cells from microbial species valuable in medical research and production (e.g., production of fuel such as ethanol, protein, and oil such as DHA).
  • the cells may also be cells from various organs, such as cells from human liver, kidney, or skin, etc, or may be blood cells.
  • the cells may also be various mature cell lines that are commercially available, such as 293 cells, COS cells.
  • the cells are those from healthy individuals; in other embodiments, the cells are those from diseased tissues of diseased individuals, such as cells from inflammatory tissues, or tumor cells.
  • the cells of interest are induced pluripotent stem cells. Cells can be those genetically engineered to have a specific function (e.g., to produce a protein of interest) or to generate a phenotype of interest. It should be understood that cells of interest include somatic cells and germ cells. In certain embodiments, the cells are specific cells in animals or humans.
  • genes of interest may be any nucleic acid sequences of interest, especially various genes or nucleic acid sequences related to diseases, or related to the production of various proteins of interest, or related to biological functions of interest.
  • genes or nucleic acid sequences of interest include, but are not limited to, nucleic acid sequences encoding various functional proteins.
  • a functional protein refers to a protein capable of achieving the physiological function of an organism, including a catalytic protein, a transport protein, an immune protein, and a regulatory protein.
  • the functional proteins include, but are not limited to: proteins involved in the occurrence, development and metastasis of diseases, proteins involved in cell differentiation, proliferation and apoptosis, proteins involved in metabolism, development-related proteins, and various medicinal targets, etc.
  • functional proteins may be antibodies, enzymes, lipoproteins, hormone-like proteins, transport and storage proteins, kinetic proteins, receptor proteins, membrane proteins, and the like.
  • genes of interest include but are not limited to RPS24, CD45, DMD, PKM, BAP1, TP53, STAT3, GANAB, ThyN1, OS9, SMN2, ⁇ -hemoglobin gene, LMNA, MDM4, Bcl2, and LRP8, etc.
  • the methods described herein include transferring the fusion protein or its expression vector and its recognized sgRNA or expression vector thereof or gDNA or expression vector thereof into the cell.
  • the corresponding sgRNA or expression vector thereof or its recognized gDNA or expression vector thereof can be transferred into the cell alone.
  • the cell inducibly expresses the fusion protein described herein after being transfered with the sgRNA or gDNA, the cell can also be incubated with an inducing agent, or the cell can be subjected to corresponding induction means (such as lighting).
  • the method herein is implemented using the fusion protein of the AID or a fragment or mutant thereof described herein and the Cas enzyme described herein, together with its recognized sgRNA.
  • transfection methods can be used to transfer into cells the fusion protein or its expression vector and/or its recognized sgRNA or expression vector thereof or gDNA or expression vector thererof.
  • the cell of interest is a prokaryotic organism such as E.coli
  • competent cells that can absorb DNAs can be harvested after the exponential growth phase and treated with the CaCl 2 method, which is well known in the art.
  • Another method is to use MgCl 2 .
  • transformation can also be carried out by electroporation.
  • the host is a eukaryote
  • the following DNA transfection methods can be used: the calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
  • the plasmid DNA-liposome complex is prepared and co-transfected into the cell together with the corresponding sgRNA or gDNA.
  • transfection kits or reagents can be used to transfer the vectors or plasmids described herein into cells of interest, such reagents include but are not limited to Lipofectamine® 2000 reagents.
  • the obtained transformants can be cultured by conventional methods to express the fusion proteins described herein. According to the used cells, the culture medium can be selected from various conventional culture media.
  • expression vectors expressing the fusion protein and sgRNA or gDNA of the present disclosure can be designed using known techniques, so that these expression vectors are suitable for expression in the cells.
  • a promoter and other related regulatory sequences that facilitate starting expression in the cell can be provided in the expression vector. These can be selected and implemented by technicians according to actual practice.
  • the site that suitable as a PAM can be found near the splice site of interest of the gene of interest, and the Cas enzyme that recognizes the PAM can be selected based on the PAM, and then the fusion protein herein containing the Cas enzyme together with its corresponding sgRNA can be designed and prepared as described herein. Therefore, the target recognition region of the sgRNA used herein usually contains the complementary sequence of the splice site(s) of the intron of interest of the gene of interest.
  • the splice site described herein has a well-known meaning in the art, including 5' splice site and 3' splice site.
  • both the 5' splice site and the 3'splice site are relative to an intron.
  • the site that can serve as a PAM is selected near the splice site of the exon/intron of interest of the gene of interest.
  • the exon or intron of interest of the gene of interest may be exon 5 of RPS, exon 5 of CD45, exon 8 or 9 of TP53 gene, exon 9 or 10 of PKM, intron 2 of BAP1 and intron 8 of TP53, etc.
  • the site that can serve as a PAM is selected near the polypyrimidine chain present within the intron upstream of the 3' splice site of the gene of interest. Therefore, the target binding region of such sgRNA contains the sequence of the polypyrimidine region of the intron of interest of the gene of interest.
  • the method herein may be a method in vitro or a method in vivo; in addition, the method herein includes a method for therapeutic purposes and a method for non-therapeutic purposes.
  • the fusion protein herein or its expression vector and its recognized sgRNA or expression vector thereof or gDNA or expression vector thereof can be transferred into the body of the subject, such as corresponding tissue cells, by methods well known in the art.
  • the subjects may be humans or various non-human animals, including various non-human model organisms commonly used in the art. Experiments in vivo should meet ethical requirements.
  • the method described herein for inducing base mutations at the splice site of a gene of interest in a cell of interest is a general RNA splicing regulation method that can be used for gene therapy.
  • a method for gene therapy comprising administering to a subject in need a therapeutically effective amount of a vector expressing the fusion protein described herein and a vector expressing the corresponding sgRNA or gDNA.
  • the therapeutically effective amount can be determined according to the age, sex, nature and severity of the disease, etc.
  • administration of a therapeutically effective amount of the vector should be sufficient to alleviate the symptoms of the disease or cure the disease.
  • the gene therapy can be used for the treatment of diseases caused by genetic mutations, and can also be used for the treatment of diseases in which symptoms of the diseases can be relieved or the diseases can be cured by regulating different splicing isoforms.
  • diseases caused by genetic mutations include but are not limited to: Duchenne myasthenia caused by mutations in the DMD gene, SMN, thalassemia caused by 647G>A mutation of ⁇ hemoglobin IVS2, familial hypercholesterolemia and premature aging caused by LMNA mutation, etc.
  • Diseases in which symptoms of the diseases can be relieved or the diseases can be cured by regulating ratio of different splicing isoforms include tumors, the splicing isoforms including but not limited to conversion of Stat3 ⁇ to Stat3 ⁇ , conversion of PKM2 to PKM1, MDM4 exon 6 skipping, selection of Bcl2 alternative splice sites, LRP8 exon 8 skipping.
  • a method for tumor therapy comprising administering to a subject in need a therapeutically effective amount of a vector expressing the fusion protein described in any embodiment herein and a vector expressing corresponding sgRNA.
  • the target binding region of the sgRNA comprises the complementary sequence of the 3' splice site of Stat3 intron 22.
  • the target binding region of sgRNA suitable for the method is shown as SEQ ID NO: 3.
  • the target binding region of the sgRNA comprises the complementary sequence of the 5' or 3' splice site of PKM intron 10.
  • the target binding region of sgRNA suitable for the method is shown as SEQ ID NO: 15 or 16.
  • a method of treating Duchenne myasthenia due to a DMD gene mutation comprising the step of administering to a subject in need a therapeutically effective amount of a vector expressing the fusion protein described herein and a vector expressing corresponding sgRNA, wherein the target binding region of the sgRNA comprises the complementary sequence of the 5' splice site of DMD exon 50.
  • the target binding region of sgRNA suitable for the method is shown as SEQ ID NO: 17 or 51.
  • tthe amino acid sequence of the fusion protein suitable for the method is shown as SEQ ID NO: 23 or 50.
  • the routes of administration for gene therapy include routes ex vivo and routes in vivo.
  • suitable backbone vectors such as adeno-associated virus vectors
  • adeno-associated virus vectors can be used to construct expression vectors expressing the fusion protein described herein and vectors expressing the sgRNA or gDNA, which can be administered to the patient in a general route, such as injection.
  • blood cells having a gene variation of the subject may be obtained, treated in vitro using the method described herein, proliferated in vitro after the the variation is eliminated, and then reinfused into the subject.
  • the methods described herein can also be used to modify pluripotent stem cells of the subject, which are reinfused into the subject to achieve therapeutic purposes.
  • fusion protein in yet another aspect of the present disclosure, is use of the fusion protein, its coding sequence and/or expression vector, and/or sgRNA and/or its expression vector according to any of the embodiments herein in the preparation of a reagent or a kit for regulating RNA splicing, in the preparation of a reagent for gene therapy, or in the preparation of a medicament for the treatment of diseases caused by genetic mutations or tumors that benefit from changes in the proportion of different splicing isoforms of functional proteins.
  • This disclosure is also directed to the fusion protein, its coding sequence and/or expression vector, and the sgRNA and/or its expression vector, according to any of the embodiments described herein, for regulating RNA splicing, gene therapy (especially for the treatment of diseases caused by genetic mutations or tumors benefiting from changes in the proportion of different splicing isoforms of functional proteins).
  • the methods described herein can effectively induce exon skipping (e.g., RPS24 exon 5, CD45 exon 5, DMD gene exon 50, 23, 51, etc.), regulate the selection of mutually exclusive exons (PKM1/PKM2, etc.), induce intron retention/inclusion (BAP1 and TP53, etc.) and induce the use of alternative splice sites (STAT3 ⁇ / ⁇ , etc.), and the like.
  • exon skipping e.g., RPS24 exon 5, CD45 exon 5, DMD gene exon 50, 23, 51, etc.
  • PPM1/PKM2, etc. regulate the selection of mutually exclusive exons
  • BAP1 and TP53, etc. induce intron retention/inclusion
  • STAT3 ⁇ / ⁇ , etc. alternative splice sites
  • the method disclosed herein is a general RNA splicing regulation method, which can be used for treatment of diseases, especially for gene therapy of the following diseases: Duchenne myasthenia caused by mutations in the DMD gene, SMN, thalassemia caused by 647G>A mutation of ⁇ hemoglobin IVS2, familial hypercholesterolemia and premature aging caused by LMNA mutation.
  • the method described herein can also achieve the treatment of tumors and other diseases by regulating the ratio of different splicing isoforms, including but not limited to inducing conversion of Stat3 ⁇ to Stat3 ⁇ , conversion of PKM2 to PKM1, MDM4 exon 6 skipping, selection of Bcl2 alternative splice sites, LRP8 exon 8 skipping, etc.
  • the AIDX-nCas9-Ugi fusion protein was used, and its expression plasmid, namely M091-AIDX- XTEN-nCas9-Ugi, was constructed according to the methods of Examples 1-3 and 14 of CN 201710451424.3 , which expressed the fusion protein of SEQ ID NO: 23, wherein, residues 1-182 is the amino acid sequence of AIDX, residues 183-198 is the amino acid sequence of linker XTEN, residues 199-1566 is the amino acid sequence of nCas9, and residues 1567-1570 and 1654-1657 are linker sequences, residues 1571-1653 is the amino acid sequence of Ugi, and residues 1658-1664 is the amino acid sequence of SV40 NLS.
  • the coding sequence of the fusion protein is shown as SEQ ID NO: 22.
  • 293T Cells were grown to 70-90% confluence before transfection.
  • plasmid DNA-liposome complexes were prepared by diluting four-folds amount of Lipofectamine® 2000 reagent in Opti-MEM® medium, and separately diluting the plasmid expressing the fusion protein described herein and the plasmid for the corresponding sgRNA in Opti-MEM® medium, then adding the diluted plasmids to the diluted Lipofectamine® 2000 reagent (1:1) and incubating for 30 minutes. The plasmid DNA-liposome complex was then transfected into 293T cells.
  • RPS24 is a constituent protein of ribosomes, and its mutation will cause congenital aplastic anemia. Exon 5 of RPS24 can be alternatively spliced to produce two isoforms with different 3' UTRs, in which liver cancer cells tend to express the isoform containing exon 5. However, its physiological function is not clear.
  • TAM technology was used to design the sgRNA (RPS24-E5-5'SS, the sequence of its target binding region is shown in SEQ ID NO: 9), and the G of the 5' splice site or 3' splice site of RPS24 exon 5 was mutated to A, regulating alternative splicing of exon 5.
  • 293T cells were transfected as described above, and gene expression, splicing and mutation were analyzed by high-throughput sequencing on the 7th day after transfection.
  • the fusion protein targeted to the 5' splice site of RPS24 exon 5 by use of the UNG inhibitor UGI in the AIDX-nCas9-Ugi fusion protein and the sgRNA.
  • the first base of intron 5 (IVS5+1) had more than 40% of G to A mutations, and the last base of exon 5 had 30% of G to A mutations, while there were other two sites on exon 5 having less than 10% of G to A mutations ( Figure 3, A ).
  • CD45 is a receptor tyrosine phosphatase, which can regulate the development and function of T lymphocytes and B lymphocytes by regulating the signaling of antigen receptors (such as TCR or BCR).
  • the CD45 gene consists of approximately 33 exons, in which exons 4, 5, and 6 encoding the extracellular regions A, B, and C of the CD45 protein can be alternatively spliced.
  • the expression pattern of the CD45 isoforms depends on the developmental stage of T cells and B cells.
  • the longest CD45 isoform (B220) containing the three selective exons is expressed on the surface of B cells.
  • the sgRNAs (CD45-E5-5'SS and CD45-E5-3'SS, the sequences of their target binding region were SEQ ID NO: 1 and 2) for the Gs at 5' splice site and 3' splice site of exon 5 in CD45 gene were designed.
  • the editing of exon 5 splice sites was performed in Raji cells, a germinal center B cell line expressing the unspliced CD45 isoform.
  • TAM technology was used to design sgRNA (TP53-E8-5'SS, the sequence of which is shown in SEQ ID NO: 7), and the G of the 3' splice site of TP53 exon 8 was mutated to A, regulating alternative splicing of exon 8 ( Figure 4 ).
  • 293T cells were transfected as described above, and gene expression, splicing and mutation were analyzed by high-throughput sequencing on the 7th day after transfection.
  • TP53-E9-3'SS TP53 exon 9
  • SEQ ID NO: 8 the target binding sequence of TP53 exon 9
  • intron-exon junctions were amplified from genomic DNA and analyzed by high-throughput sequencing.
  • TP53 splicing was analyzed by RT-PCR. The splicing junctions were amplified from cDNA and analyzed by high-throughput sequencing.
  • TAM-treated cells also activated the neuronal exon within intron 8 (4.3% of the total transcripts) ( Figure 4 , D-F).
  • the selection of alternative splice sites may occur during RNA splicing, and new protein isoforms with different physiological functions may be formed.
  • the selection of an alternative splice site on exon 23 of Stat3 will result in a truncated STAT3 ⁇ isoform lacking the C-terminal transactivation domain.
  • the full-length STAT3 ⁇ can promote tumorigenesis, while STAT3 ⁇ has dominant negative effect, inhibiting STAT3 ⁇ function and promoting tumor cell apoptosis.
  • inducing STAT3 ⁇ expression can inhibit cell survival more effectively compared to knocking out STAT3 expression, indicating that inducing STAT3 ⁇ expression can be used as a tumor therapy.
  • 293T cells were transfected with AIDx-nCas9-Ugi and the sgRNA targeting Stat3 exon 23 (STAT3-E23-3'SS, its target binding region is shown in SEQ ID NO: 3) or the sgRNA targeting AAVS1 (Ctrl).
  • Intron-exon junctions were amplified from DNA (top 2 panels) or cDNA (bottom 2 panels) and analyzed by high-throughput sequencing.
  • TAM and sgRNA were expressed in 293T cells using the method described above, and more than 50% of the Gs at 3' splice site were mutated to As ( Figure 5, B ). Results show that TAM enhanced the use of the distal 3'SS in Stat3 exon 23 ( Figure 5, C ).
  • TAM technology can overcome the defects of conventional double sgRNA splicing methods, accurately destroy selective splice sites, and regulate the selection of alternative splice sites.
  • mutually exclusive exon is another major type of alternative splicing, in which mutually exclusive exons can be selectively included in different transcripts to produce proteins with different functions.
  • Pyruvate kinase PKM
  • exons 9 and 10 of PKM can be selectively included to produce two isoforms PKM1 and PKM2, wherein PKM1 containing exon 9 but not exon 10 is mainly expressed in adult tissues, while PKM2 containing exon 10 but not exon 9 is mainly expressed in embryonic stem cells and tumor cells. Because PKM2 is related to tumorigenesis, it is hoped that TAM technology can switch the PKM splicing mode of tumor cells from PKM2 to PKM1.
  • Figure 6(A) shows a schematic diagram of TAM switching PKM2 to PKM1 in C2C12 cells.
  • exon 10 of the PKM gene rather than exon 9 was spliced to produce PKM2, whose cDNA was recognized by the restriction enzyme PstI; in the bottom panel, TAM converted the GT dinucleotide to AT at the 5'SS of exon 10. Therefore, exon 9 instead of exon 10 was spliced to produce PKM1, whose cDNA was recognized by the restriction enzyme NcoI.
  • PKM-3'SS-E10 or PKM-5'SS-E10 the sequence of their target binding region is SEQ ID NO: 15 or 16, respectively
  • PKM3'SS-E10 or PKM-5'SS-E10 the sequence of their target binding region is SEQ ID NO: 15 or 16, respectively
  • intron 10 were designed and transferred into C2C12 cells to mutate the G to A ( Figure 6 , C, D). It was found that in the muscle cells differentiated from C2C12, PKM2 expression was significantly down-regulated and PKM1 expression was up-regulated ( Figure 6 , B, E, F). Similarly, in undifferentiated C2C12 cells, PKM2 expression was significantly down-regulated and PKM1 expression was up- regulated ( Figure 6 , G, H).
  • the G could be mutated to A, while PKM1 expression level was down-regulated ( Figure 7 ) and PKM2 expression was up-regulated. This further proved that the mutation of the splice site(s) can change the selection of the splice site(s) of mutually exclusive exons.
  • Intron retention is another type of alternative splicing, and recent studies have shown that intron retention occurs in many human diseases including tumors. We demonstrated that the use of TAM and sgRNA to disrupt the splice site(s) of a corresponding intron can specifically induce intron retention.
  • BAP1 is a histone deubiquitinase, and its second intron is retained in some tumors, causing a decrease in BAP1 expression.
  • the second intron of BAP1 may be spliced in an intron-defined manner, wherein the 5'SS is paired with the downstream 3'SS.
  • the G is converted to A, and U1 recognizes U1 RNP at 5'SS and destroys the intron definition, resulting in the inclusion of the intron.
  • This experiment used TAM to mutate G at the 5' splice site of intron 2 of BAP1, the schematic diagram of which is shown in Figure 8(A) .
  • SgRNA (BAP1-E2-5'SS, its target binding region is shown in SEQ ID NO: 5) targeting the 5' splice site of intron 2 was designed.
  • 293T cells were transfected with the expression plasmid of AIDx-nCas9-Ugi and the expression plasmid of the sgRNA targeting AAVS1 (Ctrl) or BAP1 intron 2.
  • BAP1 mRNA splicing was analyzed by RT-PCR ( Figure 8, B ) or isoform-specific real-time PCR ( Figure 8, C ). The results show that more than 70% of Gs were mutated to As ( Figure 8, D ).
  • TAM technology can also be used to edit other splicing regulatory elements. Because changes in introns do not affect the sequences for gene expression, we focused on the editing of splicing regulatory elements of intron.
  • a polypyrimidine chain consisting of cytosine (C) and thymine (T) is present upstream of the 3' splice site. This experiment proved that the C in the polypyrimidine chain can be mutated to T by TAM and the corresponding sgRNA, therefore enhancing the strength of the 3' splice site and promoting the inclusion of downstream exons.
  • 293T cells were transfected with the expression plasmid of AIDx-nCas9-Ugi and the expression plasmid of sgRNA targeting AAVS1 (Ctrl) or sgRNA targeting polypyrimidine nucleosides of the fifth exon in RPS24 (RPS24-E5-PPT, its target binding region is shown as SEQ ID NO: 10).
  • sgRNA targeting regions were amplified from genomic DNA and analyzed by high-throughput sequencing with over 8000x coverage. The results show that more than 50% of the Cs in the polypyrimidine chain were mutated to Ts. It was found that the inclusion rate of exon 5 increased ( Figure 11 , B, C). After sorting, two single-cell clones containing complete C to T mutations were obtained, and their inclusion rate of exon 5 was increased by 8-fold and 5-fold, respectively ( Figure 11, E ).
  • 293T cells were transfected with the expression plasmid of AIDx-nCas9-Ugi and the expression plasmid of control sgRNA (Ctrl) or sgRNA targeting PPT of exon 6 in GANAB (GANAB-E6-PPT, its target binding region is shown as SEQ ID NO: 4).
  • sgRNA targeting regions were amplified from genomic DNA and analyzed by high-throughput sequencing with over 8000x coverage. The results are shown in Figure 10 (B-E), wherein multiple Cs were induced to mutate to Ts, with the highest being IVS5-6C, in which more than 70% of the Cs were mutated to Ts.
  • TAM technology can restore DMD protein expression in human iPS cells and mdx mouse models (C2C12 and iPS)
  • Duchenne muscular dystrophy is a muscular dystrophy disease. There is one case for every 4,000 men in the United States. The heritable mutation of the patient's DMD gene leads to the change of the gene's open reading frame or the formation of immature codons, resulting in dystrophin defects in skeletal muscle and the occurrence of the disease. Compared with the mutated DMD gene, the truncated dystrophin retains partial function, resulting in Becker muscular dystrophy with mild symptom. Therefore, some studies have used antisense oligonucleotides or double sgRNA-mediated CRISPR technology to skip some exons, so that to restore the open reading frame of DMD and promote the expression of dystrophin.
  • iPS cells of a DMD patient lacking exon 51 is used in this experiment.
  • the open reading frame of dystrophin protein was restored ( Figure 12 ).
  • the iPSCs from the patient were transfected with the expression plasmid of sgRNA (the sequence of the target binding region is shown in SEQ ID NO: 17) and the expression plasmid of AIDx-nCas9-Ugi.
  • High-throughput sequencing shows that it can induce more than 12% of G>A mutations ( Figure 12, B ), and then a monoclonal cell having complete G>A mutations were obtained ( Figure 12B ). Then the iPSCs were differentiated into cardiomyocytes and it was found that the TAM-edited cells had exon 50 skipping ( Figure 12C, D ). Further, western bloting shows that the expression of the dystrophin protein was restored in the TAM-repaired cells ( Figure 12, E ).
  • skipping of DMD exon 50 was induced by AIDx-saCas9 (KKH, nickase)-Ugi (coding sequence: SEQ ID NO: 49, amino acid sequence: SEQ ID NO: 50) and the corresponding sgRNA sequence (the sequence is shown in SEQ ID NO: 51, and its backbone sequence is shown in SEQ ID NO: 52).
  • sequence involved in this disclosure is as follows: Sequence No. Name Sequence No. Name 1 CD45-E5-5'SS 27 dcas9-aidm 2 CD45-E5-3'SS 28 AIDx-XTEN-dCas9 3 STAT3-E23-3'SS 29 AIDx-XTEN-dCas9 4 GANAB-E6-PPT 30 dCas9-XTEN-AID P182X K10E T82I E156G 5 BAP1-E2-5'SS 31 dCas9-XTEN-AID P182X K10E T82I E156G 6 BAP1-E3-3'SS 32 ncas9-P182x 7 TP53-E8-5'SS 33 ncas9-P182x 8 TP53-E9-3'SS 34 PAM sequence 9 RPS24-E5-5'SS 35 10 RPS24-E5-PPT 36 11 OS9-E13-PPT 37 12 THYN1-E6-PPT 38 13 PKM-3'

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